Normal bone formation occurs during development and repair after injury and remodeling occurs in adult life to preserve the integrity of the skeleton. Bone formation and remodeling generally involve bone resorption by osteoclasts and bone formation by osteoblasts, which are regulated by growth factors. Thus, any interference between the balance in cell differentiation, bone formation and bone resorption can affect bone formation, repair and homeostasis.
Noggin is a protein which is present and highly conserved in both protostome and deuterstome phyla and which interacts with bone mophogenic proteins (“BMPs”) to affect embryonic orientation and tissue layer formation in early development. (Reddi, A. H. (2001). Interplay between bone morphogenetic proteins and cognate binding proteins in bone and cartilage development: noggin, chordin and DAN. Arthritis Res. 3:1-5. Zimmerman; L. B., et al. (1996) The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86:599-606.) It is a 33 kD glycoprotein which is secreted as a 64 kD homodimer and was shown to perform the functions of the active principle of the Spemann organizer in Xenopus in that it induced neural tissue from dorsal ectoderm and changed the specification of lateral mesoderm from ventral (blood, mesenchyme) to dorsal (muscle, heart, pronephros) fates during gastrulation (Smith, W. C., et al. (1993). Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm. Nature 361:547-549. Lamb, T. M., et al. (1993) Neural induction by the secreted polypeptide noggin. Science 262:713-718). BMP-4 had been demonstrated to act as a ventralizing factor in Xenopus development (Dale, L., et al. (1992) Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development. Development 115:573-585. Jones, C. M., et al. (1992) DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction. Development 115:639-647) and to inhibit neural induction (Wilson, P. A., and Hemmati-Brivanlou, A. (1995) Induction of epidermis and inhibition of neural fate by BMP 4. Nature 376:331-333) suggesting that noggin may function by interacting with BMP-4. Subsequent studies demonstrated that noggin avidly binds BMP-4 and inhibits its activity by blocking binding of BMP-4 to cellular receptors (Zimmerman, L. B., et al. (1996) The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86:599-606. Groppe, J., Greenwald, J., et al. (2002) Structural basis of BMP signaling inhibition by the cystein knot protein noggin. Nature 420:636-642).
Noggin performs important regulatory functions in the vertebrate skeleton (Brunet, L. J., et al. (1998) Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280:1455-1457. Devlin, R. D., et al. (2003) Skeletal over expression of noggin results in osteopenia and reduced bone formation. Endocrinology 144:1972-1978. Nakamura, Y., Wakitani, S., Nakayama, J., et al. (2003) Temporal and spatial expression profiles of BMP receptors and noggin during bmp-2-induced ectopic bone formation. J. Bone Min. Res. 18:1854-1862. Stottmann, R. W., et al. (2001) The BMP antagonists chordin and noggin have essential but redundant roles in mouse mandibular outgrowth. Dev. Biol. 240:457-473). Noggin reduced BMP-4-induced expression of alkaline phosphatase in W-20-17 mouse bone marrow stromal cells, which were isolated on the basis of manifesting an osteoblast-like phenotype after treatment with rhBMP-2 (Theis, R. S., et al. (1992) Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130:1318-1324). Noggin was shown to be expressed in condensing cartilage in mice, and mice genetically altered to lack noggin were found to have shorter but thicker limbs and multiple synarthroses (Brunet, L. J., et al. (1998) Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. Science 280:1455-1457). Human mutations of the noggin gene have been shown to be associated with fibrodysplasia ossificans progressiva and synarthrosis syndromes (Semonin, O., et al. (2001) Identification of three novel mutations of the noggin gene in patients with fibrodysplasia ossificans progressiva. Am. J. Med. Genet. 102:314-317. Gong, Y., et al. (1999) Heterozygous mutations in the gene encoding noggin affect human joint morphogenesis. Nature Genet. 21:302-304).
Delivery of modified or wild-type noggin in a bone chamber reduced in-growth of new bone at four weeks in rats (Aspenberg, P., et al. (2001) The bone morphogenetic proteins antagonist noggin inhibits membranous ossification. J. Bone Mineral. Res. 16:497-500). Transgenic mice which over-expressed noggin under the control of the osteocalcin promoter exhibited diminished bone mineral density, reduced trabecular number and volume, reduced bone formation rate, but normal trabecular osteoclast surface with reduced osteoclast number (Devlin, R. D., et al. (2003) Skeletal over expression of noggin results in osteopenia and reduced bone formation. Endocrinology 144:1972-1978). Noggin delivered through a retroviral vector inhibited ectopic bone formation induced by demineralized bone matrix or vector-delivered BMP-4 (Hannallah, D., et al. (2004) Retroviral delivery of noggin inhibits the formation of heterotopic ossification induced by BMP-4, demineralized bone matrix, and trauma in an animal model. J. Bone Joint Surg. 86:80-91).
Demineralized bone matrix (DBM) and native bone morphogenetic protein (nBMP) are partially purified extracts of non-collagenous bone proteins that contain at least one endogenous BMP. BMP's belong to a family of growth factors described as bone morphogenic protein/non-collagenous protein (BMP/NCP or BMPs). Specifically BMP-2 and BMP-4 have been isolated therefrom, and are in the TGF-β family of proteins. However, DBM and nBMP also include enhancers and inhibitors of BMP activity.
For example, recently BBP has been disclosed as enhancing ossification caused by recombinant BMP. Further, BBP as used with BMP in vivo causes osteogenesis to occur faster, to a greater extent and with smaller amounts of rhBMP-2.
DBM and nBMP exhibit osteogenic potential, and can be used to enhance bone healing in a number of clinical situations. (Urist, M. R. (1965) Bone: Formation by autoinduction. Science 150:893-899; Reddi, A. H. and Huggins, C. (1972) Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc. Natl. Acad. Sci. U.S.A. 69:1601-31605 and Urist, M. R., et al. (1984) Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc. Natl. Acad. Sci. U.S.A. 81:371-375. (Urist, M. R. Emerging Concepts of Bone Morphogenetic Protein. (1991) In: Fundamentals of Bone Growth: Methodology and Applications, A. D. Dixon, B. G. Sarnat, and D. A. N. Hoyte (eds.), pp. 189-198. (C.R.C. Press, Boston)). Because of the number of BMP-related proteins found in DBM and the complexity of their regulatory functions, it is likely that partially purified mixtures such as DBM and nBMP contain a number of molecules that affect the availability and activity of the BMPs. While recombinant BMPs are available, the cost of using minimally effective dosages of BMP has been a limiting factor in clinical use. Therefore, DBM and nBMP having increased osteogenic activity are desired.
Safe, effective and affordable compositions, devices and methods are desired to treat bone disorders (such as osteoporosis), bone injury (such as fracture healing of flat (e.g., membranous) and long (e.g., endochondral) bones, non-union fractures and reconstructive surgery), sites of knee/hip/joint repair or replacement surgery treating periodontitis, periodontal regeneration, alveolar ridge augmentation for tooth implant reconstruction, for example.
Further, the use of compositions and methods in combination with other methods for enhancing osteogenesis are desirable.
It may be desirable to improve DBM and nBMP by adding activators and substances which improve the kinetics or overall osteogenic potential of BMP action or by removing inhibitors of BMP action, for example.
Further, methods for removing inhibitors of BMPs, while not adversely effecting the osteogenic activity of BMPs in DBM (such as BMP-2, -4 and -7 (known as “osteogenin”), are desirable. Also desirable are DBM and/or rBMP which produces more bone per volume or weight than native DBM or nBMP obtained from non-processed DBM. Finally, methods which do not adversely effect the osteogenic potential of DBM are desirable.